PROJECT SUMMARY/ABSTRACT Cardiovascular disease is the leading cause of death and stroke in developed countries. Long-term outcomes following treatments for severe atherosclerosis are limited by the development of neointimal hyperplasia leading to restenosis of the vessel. Thus, there is a need for better and more durable therapies to prevent restenosis after vascular interventions. Nitric oxide (NO) is a potent inhibitor of neointimal hyperplasia. While much is known about how NO regulates the arterial injury response, little is known about how NO may regulate progenitor cells in this process. Preliminary data from our laboratory have shown that progenitor cells that reside in the adventitia, namely Sca1+ progenitor cells, increase in the intima and media following arterial injury. NO prevents this increase, while also redirecting differentiation of these cells toward endothelial cells. While Sca1+ progenitor cells have the capacity to differentiate into multiple cell types, including vascular smooth muscle cells and endothelial cells, their functional role in the development of neointimal hyperplasia is unknown. Thus, the goal of this proposal is to explore these unknowns. We hypothesize that resident adventitial Sca1+ progenitor cells differentiate into vascular smooth muscle cells and contribute to the development of neointimal hyperplasia following arterial injury, but that NO redirects differentiation of the resident Sca1+ cells toward an endothelial cell lineage, thereby limiting the development of neointimal hyperplasia. To investigate this hypothesis, our Specific Aims are: 1) to examine the effect of nitric oxide on Sca1+ vascular progenitor cells in vitro, by exposing cultured Sca1+ progenitor cells to differentiation media with and without NO and quantifying smooth muscle and endothelial cell differentiation. 2) To investigate the migration and phenotypic fate of Sca1+ vascular progenitor cells in response to injury NO exposure. Specifically, we will track the fate of Sca1+ cells after vascular injury by permanently labeling Sca1+ cells with green fluorescent protein using a Cre-lox mouse model. Mice will then undergo femoral artery wire injury NO exposure in order to study the behavior and fate of Sca1+ progenitor cells after vascular injury. 3) To determine the functional role of Sca1+ vascular progenitor cells in the development of neointimal hyperplasia. Specifically, we will study the effect of loss of Sca1+ cells using a genetically engineered mouse that will selectively and temporally eliminate Sca1+ progenitor cells by expressing diphtheria toxin once the Sca1 gene is activated. Femoral artery wire injury will be performed on these mice NO. Successful completion of the studies described in this proposal will elucidate the role of Sca1+ progenitor cells in the development of neointimal hyperplasia and restenosis. This research may lead to the development of novel therapeutics that regulate Sca1+ progenitor cells following vascular procedures to prevent restenosis. Execution of these studies will help me to gain the skills necessary to develop as an independent physician-scientist.